The Epstein-Barr virus nuclear antigen (EBNA) has received much attention since its expression in certain human cells correlated with malignant transformation as opposed to viral replication. We have recently identified two classes of EBNA. Class I EBNA which has been studied by several laboratories is extracted from the nuclear chromatin using solutions of 0.0 to 1 M GuHC1 or 0.3 to 1.50 M NaCl. Class II EBNA, tightly bound to chromatin, is extracted with 3 to 4 M GuHCl. Comparisons of the soluble "S" antigen, the Class I and Class II EBNA by isoelectric focusing and molecular sieve chromatography suggest that the "S" antigen and Class I EBNA are similar. Class II EBNA, however, appears to be greatly different. The Class II EBNA has a very high affinity for DNA, focuses with an apparent pI of 9.0 and chromatographs as two classes of basic proteins with molecular weights of 33,000 and 64,000 making them far too large to be histones. This project proposes to purify and more thoroughly characterize this new, tightly bound class of EBNA. A variety of techniques to fractionate these hydrophobic chromosomal proteins have been developed in or adapted by this laboratory for purificaton of this antigen; these include preparative isolelectric focusing in 6 M urea, molecular sieve chromatography, ion exchange chromatography in 4 M urea, and adsorption chromatography. The chemical composition, exact size, and specificity of Class II EBNA for DNA sequence (single or double strand) will be determined. This class II EBNA will be compared to the soluble CF antigen and the Class I EBNA using the above methods. Attempts to prepare antisera against this highly purified Class II EBNA will be made to better detect and characterize this antigen(s).